SARS-CoV-2 variants and mutational patterns: relationship with risk of ventilator-associated pneumonia in critically ill COVID-19 patients in the era of dexamethasone

We aimed to explore the relationships between specific viral mutations/mutational patterns and ventilator-associated pneumonia (VAP) occurrence in COVID-19 patients admitted in intensive care units between October 1, 2020, and May 30, 2021. Full-length SARS-CoV-2 genomes were sequenced by means of next-generation sequencing. In this prospective multicentre cohort study, 259 patients were included. 222 patients (47%) had been infected with pre-existing ancestral variants, 116 (45%) with variant α, and 21 (8%) with other variants. 153 patients (59%) developed at least one VAP. There was no significant relationship between VAP occurrence and a specific SARS CoV-2 lineage/sublineage or mutational pattern.

Current data on ventilator-associated pneumonia (VAP) complicating COVID-19-associated acute respiratory distress syndrome (ARDS) have been mainly obtained during the first wave of COVID-19 1 . Yet, bacterial superinfection may be affected by the nature of the infecting viral variants and the widespread use of corticosteroids 2 . There is scarce information in the literature on the rate of superinfections in COVID-19 patients, especially of VAP, according to variant types in the dexamethasone era. Potential explanations for the high incidence of VAP in COVID-19 patients include the long duration of invasive mechanical ventilation support, the high incidence of ARDS, and the administration of immune-suppressive treatment. Specific risk factors for VAP, including SARS-CoV-2-related pulmonary lesions and bacteria-virus interaction in lung microbiota, might also play a role in VAP pathogenesis. As the upper respiratory tract microbiome composition significantly changed as COVID-19 severity increased 3 , we hypothesize that specific SARS-CoV-2 mutations, either in the spike protein or in another viral protein, could have an impact on VAP prevalence.
In this study, we aimed to explore the relationships between SARS-CoV-2 variant lineage/sublineage and specific viral mutations/mutational patterns with VAP occurrence in COVID-19 patients in the dexamethasone era.

Patients and methods
This is a prospective multicentre observational cohort study. Patients admitted between October 1, 2020 (week 40/20) and May 30, 2021 (week 21/21) in one of the 11 participating ICUs from Greater Paris area hospitals included in the ANTICOV study (NCT04733105) were eligible for study inclusion. Inclusion criteria were as follows: age ≥ 18 years, SARS-CoV-2 infection confirmed by a positive reverse transcriptase-polymerase chain reaction (RT-PCR), patient admitted in the ICU who required mechanical ventilation more than 48 h for acute respiratory failure. Patients with a low viral load (PCR cycle threshold (Ct) > 32) in nasopharyngeal samples were not included in the study.
The primary endpoint was the difference in prevalence of the first VAP according to SARS-CoV-2 variants.
Ventilator associated pneumonia definition. All VAP episodes prospectively diagnosed by attending physicians were evaluated and retained for analyses, provided that they fulfilled the following criteria (the definition of VAP was based on current guidelines 4 ): (1) new or progressive persistent pulmonary infiltrates on chest X-ray; combined with (2)  Ethical approval and consent to participate. The study was approved by the Comité de Protection des Personnes Nord-Ouest IV (N° EudraCT/ID-RCB: 2020-A03009-30). Informed consent was obtained from all patients or their relatives. All study procedures and processes were conducted in accordance with the Declaration of Helsinki and with the ethical standards of the institutional and/or research committee. , γ (P.1) (n = 2, 0.5%) and other variants of interest (n = 12, 2.9%). One hundred and fifty-three patients (59%) developed at least one VAP episode. During the study period, variant α was the dominant VOC in the Greater Paris area. Thus, we first assessed whether patients infected with variant α had a higher risk of developing VAP than others (merging patients infected with preexisting variants and other variants) during the ICU stay. The Fine and Gray model showed that the probability of VAP was not significantly different in the two groups [sub-hazard ratio = 1.26 (0.62-2.54), p = 0.53, Fig. 1] after adjusting for weaning and death as competing events.
Pre-existing and emerging variants (VOC or VOI) are characterized by multiple lineage-specific deletions and amino acid substitutions in their viral genomes. The Spike glycoprotein undergoes evolutive convergence at several amino-acid signature sites found in VOC and VOI circulating at the time of the study period. These sites were selected a priori for in-depth analysis. We found no significant relationship between VAP occurrence and any specific mutation at these sites ( Table 2). These results persisted after adjusting for age, sex, antibiotic at admission and SAPS II (Table 3). Although one mutation (A67V) was found to be significantly associated with www.nature.com/scientificreports/ the risk of VAP (p < 0.05), the number of patients harboring this mutation was considered too small (n = 1) to draw clinically relevant conclusions.

Discussion
We previously failed to find a significant relationship between variant lineages, including variant α, pre-existing and other variants, and mortality 7 . The main results of this study show the lack of statistically significant relationship between variant lineages or the presence of any of 17 relevant spike substitutions and/or deletions selected a priori and VAP occurrence. These findings suggest that the occurrence of VAP is not related to the virological nature of the infecting agent, but to non-virological causes.
The full-length SARS-CoV-2 genomes of 413 critically ill COVID-19 patients from 11 ICUs, who were predominantly infected with pre-existing and α (B.1.1.7) variants, were sequenced and the relationship between viral sequences and VAP occurrence was studied. The main results of this study show the lack of statistically significant relationship between variant lineages or the presence of any of 17 relevant spike substitutions and/or deletions selected a priori and VAP occurrence. Previous large-scale data have suggested that patients infected with variant α (B.1.1.7), identified using the SGTF proxy, had a higher risk of dying [8][9][10] . Yet, no specific comorbidity or clinical conditions was shown to interact with variant types in the previous studies. Interestingly Together, our results indicate that, in patients with the most severe forms of COVID-19 who required ICU admission, there was no particular mutational pattern associated with VAP. Therefore, the occurrence of VAP is not related to the virological nature of the infecting agent, but to other causes.
Limitations and strengths of the study. We acknowledge that our study has some limitations. In this cohort, included patients were predominantly infected with pre-existing and α (B.1.1.7) variants, while the inclusion period ended before the emergence of the δ and ο variants, precluding the generalizability of our findings to these more recent variants. The number of patients included was limited and thus the statistical power may have been too weak to show between-group differences. Yet, there was no clear trend regarding associations between variant groups/mutations and VAP, suggesting that increasing the number of patients in the cohort would not have changed the results.
Our study also has strengths, including the constitution of a prospective multicenter cohort of well-phenotyped critically ill patients, and the fact that we performed full-length SARS-CoV-2 genome sequencing in a large number of patients. The start of inclusion of the present study was October 1, 2020, which corresponded in France to the beginning of the second COVID-19 wave, a period when ICU admission policies, ICU patient load and demand, and COVID-19 management strategies were more homogeneous among centres than during the first COVID-19 wave. The high incidence of VAP described in our study is in line with that reported in previous studies 1,12 .

Conclusions
We found no association between the variant status or any mutational pattern in SARS CoV-2 viral genes and VAP, indicating that the occurrence of VAP is related to non-virological causes.

Data availability
The datasets supporting the conclusions are included within the article. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.  Table 3. Multivariable Fine and Gray model analysis on risk of ventilator-acquired pneumonia. SHR subhazard ratio, CI 95% 95% confidence interval; a pre-existing variant or other variants.

Mutations
Missing data SHR CI 95%